title: Proteomic assessment of a cell model of spinal muscular atrophy.
abstract: BACKGROUND
  Deletion or mutation(s) of the survival motor neuron 1 (SMN1) gene causes spinal muscular atrophy (SMA), a neuromuscular disease characterized by spinal motor neuron death and muscle paralysis. Complete loss of the SMN protein is embryonically lethal, yet reduced levels of this protein result in selective death of motor neurons. Why motor neurons are specifically targeted by SMN deficiency remains to be determined. In this study, embryonic stem (ES) cells derived from a severe SMA mouse model were differentiated into motor neurons in vitro by addition of retinoic acid and sonic hedgehog agonist. Proteomic and western blot analyses were used to probe protein expression alterations in this cell-culture model of SMA that could be relevant to the disease.
  
  
  RESULTS
  When ES cells were primed with Noggin/fibroblast growth factors (bFGF and FGF-8) in a more robust neural differentiation medium for 2 days before differentiation induction, the efficiency of in vitro motor neuron differentiation was improved from ~25% to ~50%. The differentiated ES cells expressed a pan-neuronal marker (neurofilament) and motor neuron markers (Hb9, Islet-1, and ChAT). Even though SMN-deficient ES cells had marked reduced levels of SMN (~20% of that in control ES cells), the morphology and differentiation efficiency for these cells are comparable to those for control samples. However, proteomics in conjunction with western blot analyses revealed 6 down-regulated and 14 up-regulated proteins with most of them involved in energy metabolism, cell stress-response, protein degradation, and cytoskeleton stability. Some of these activated cellular pathways showed specificity for either undifferentiated or differentiated cells. Increased p21 protein expression indicated that SMA ES cells were responding to cellular stress. Up-regulation of p21 was confirmed in spinal cord tissues from the same SMA mouse model from which the ES cells were derived.
  
  
  CONCLUSIONS
  SMN-deficient ES cells provide a cell-culture model for SMA. SMN deficiency activates cellular stress pathways, causing a dysregulation of energy metabolism, protein degradation, and cytoskeleton stability.
authors.affiliation: Department of Biological Science, University of Delaware, Newark, DE, USA.
authors.forename: Chia-Yen
authors.initials: CY
authors.lastname: Wu
authors.forename: Dosh
authors.initials: D
authors.lastname: Whye
authors.forename: Lisa
authors.initials: L
authors.lastname: Glazewski
authors.forename: Leila
authors.initials: L
authors.lastname: Choe
authors.forename: Douglas
authors.initials: D
authors.lastname: Kerr
authors.forename: Kelvin H
authors.initials: KH
authors.lastname: Lee
authors.forename: Robert W
authors.initials: RW
authors.lastname: Mason
authors.forename: Wenlan
authors.initials: W
authors.lastname: Wang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D004195:Disease Models, Animal; D004622:Embryo, Mammalian; D051524:Fibroblast Growth Factor 8 / Q000494:pharmacology; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009046:Motor Neurons / Q000378:metabolism*; D009134:Muscular Atrophy, Spinal / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013116:Spinal Cord / Q000473:pathology*; D013234:Stem Cells / Q000187:drug effects / Q000378:metabolism; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; C496457:Fgf8 protein, mouse; D009419:Nerve Tissue Proteins; D020543:Proteome; D055533:Survival of Motor Neuron 1 Protein; D049452:Green Fluorescent Proteins; D051524:Fibroblast Growth Factor 8; D005346:Fibroblast Growth Factors